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quantity of each but not the relative abundance
as is measured in the discovery phase. Chang-
ing from relative to absolute measurement is
challenging, as it requires validation of the
analytical method used in the biomarker verifi-
biomarker candidates identi
ed within the
discovery stage. Once the biomarkers are vali-
dated, the effort can be devoted to develop
assays that can actually be used in clinical
setting. Currently, enzyme-linked immunosor-
bent assay (ELISA) is the accepted standard
assay for biomarker veri
-
cation phase. Essentially, the veri
cation phase
requires the development of quantitative assays
that can quantify targets already de
cation and validation.
However, even though ELISA does offer high
sensitivity in the pg/ml range, developing
individual ELISA assays for a large number
of potential biomarkers at
ned in the
discovery phase accurately, reliably, and repro-
ducibly across many samples
in a high-
throughput fashion. Veri
cation studies must
be very carefully designed and cannot simply
be an analysis of more of the same samples
used in the discovery phase. Similar to valida-
tion, the cohorts of samples should be increased
to include not only samples obtained from
disease-affected and healthy donors but also
those with phenotypically similar diseases and
a broad range of individuals within the popula-
tion to provide the speci
cation
stage is very time consuming, expensive and
unnecessary.
Recently, quantitation assays based on
selected reaction monitoring (SRM) MS in combi-
nation with stable isotope-labeled internal stan-
dards have been extensively investigated as an
alternative to ELISAs for protein biomarker veri-
the veri
fication purposes.
3
e
9
SRM is amass spectrometry
technique that targets prede
city needed as
biomarkers. For example, individuals of both
genders, broad age range, different races and
ethnic backgrounds, pre- and postmenopausal
women, and so on should all be included to
get a statistically empowered measurement
of the concentration range of the potential
biomarkers being targeted. The types of
samples that are used are almost always serum
or plasma, owing to their availability as well as
the hypothesis that their molecular content
represents the physiological and pathological
state of the whole human body. Other biolog-
ical samples such as urine and cerebrospinal
ned analytes with
known fragmentation patterns. Fortunately,
many of the techniques required to develop
biomarker veri
cation studies have been well
established and widely utilized for small mole-
cule analysis developed over a decade ago.
10,11
The capability of this targeted MS-based
approach to monitor multiple peptide transitions
in parallel (therefore also referred to as multiple
reaction monitoring
[MRM]
in
literature
frequently) offers both the high speci
city and
the throughput needed for veri
cation of the
large numbers of biomarker candidates proposed
in the biomarker discovery stage. The principles
as well as the factors affecting the performance
of the targeted MS-based approach for peptide
quantitation will be discussed in this chapter.
This chapter is an update and extension of our
previous article
12
on the same subject with recent
progress of the application of the targeted MS
approach for biomarker veri
fluid (CSF) are also utilized. Although the
discovery phase continues to generate many
potential biomarkers,
cation phase
has become one of the major bottlenecks for
biomarker translation from the bench side to
the bedside.
Targeting speci
the veri
c biomarkers enables abso-
lute quantitation measurements of each mole-
cule to be recorded with greater accuracy and
precision than afforded in the discovery phase.
High-throughput multiplexed assays must be
developed to screen and verify a large number
cation. Sample
preparation strategies proposed to improve
sensitivity when dealing with commonly used
sample types such as plasma, software solutions
developed to facilitate the automation of both
the experimental design and data processing as
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